The ultimate goal in designing new immunomodulatory therapies for transplantation is the induction and maintenance of a state of donor-specific immunologic tolerance. The main goal of the project is to study the role of novel T cell costimulatory pathways in regulating alloimmune responses in vivo. T cell costimulatory blockade directed at the CD28-B7 and/or CD154-CD40 pathways, while effective in some rodent models, may not be effective in reproducibly inducing tolerance in murine transplant models and in primates. We hypothesize that this may be due to several mechanisms including the functions of other T cell costimulatory pathways, and/or B7/CD154 costimulation blockade resistant CD8+ alloreactive T cells or memory T cells. In this project we will focus on studying the functions of the ICOS-B7h, CD 134-CD134L (OX40-OX40L) and CD27 -CD70 T cell costimulatory pathways because of their established unique functions in immune responses. We have a number of unique and important tools that will enable us to dissect the functions, mechanisms, and interactions of these novel costimulatory pathways in regulating alloimmune responses in vivo. We have available gene knockout animals (ICOS and CD134L), a transgenic animal that constitutively expresses ICOS on T cells (ICOSc), and CD8+ and CD4+ TCR transgenic mice that are alloreactive to specific class I and class II MHC antigens, respectively. We also have monoclonal antibodies and fusion proteins directed at the newly identified T cell costimulatory molecules (ICOSL/B7h, CD134L, CD70). We plan to study the functions of the three novel T cell costimulatory pathways and dissect their interactions with the CD28/CTLA4-B7- 1/B7-2 and CD154-CD40 pathways, with the goal of achieving and defining the mechanisms of transplantation tolerance in murine vascularized cardiac and skin (as a stringent model) transplantation models. Understanding the functions of the new T cell costimulatory pathways in alloimmune responses is important for the progress of the studies planned in project two focused at investigating the fate of T cells during alloimmune responses as a determinant of tolerance, and the studies of project three aimed at understanding the functions of regulatory T cells in transplantation tolerance. Our work should help develop novel strategies to induce transplantation tolerance to translate to primates and ultimately humans.